VLA-4 (also referred to as .alpha..sub.4 .beta..sub.1 integrin and CD49d/CD29), first identified by Hemler and Takada (Hemler and Takada, European Patent Application, Publication No. 330, 506, published Aug. 30, 1989) is a member of the .beta..sub.1 integrin family of cell surface receptors, each of which comprises two subunits, an .alpha..sub.4 chain and a .beta..sub.1 chain. There are at least nine .beta..sub.1 integrins, all sharing the same .beta..sub.1 chain and each having a distinct .alpha. chain. These nine receptors all bind a different complement of the various cell matrix molecules such as fibronectin, laminin and collagen. VLA-4, for example, binds to fibronectin. VLA-4 is unique among .beta..sub.1 integrins in that it also binds non-matrix molecules that are expressed by endothelial and other cells. These non-matrix molecules include VCAM-1, which is expressed on cytokine-activated human umbilical vein endothelial cells in culture. Distinct epitopes of VLA-4 are responsible for fibronectin and VCAM-1 binding activities and each activity has been shown to be inhibited independently (Elices, et al., Cell, 60:577-584 (1990)).
Intercellular adhesion mediated by VLA-4 and other cell surface receptors is associated with a number of inflammatory responses. At the site of an injury or other inflammatory stimulus, activated vascular endothelial cells express molecules that are adhesive for leukocytes. The mechanics of leukocyte adhesion to endothelial cells involves, in part, the recognition and binding of cell surface receptors on leukocytes to the corresponding cell surface molecules on endothelial cells. Once bound, the leukocytes migrate across the blood vessel wall to enter the injured site and release chemical mediators to combat infection. For reviews of adhesion receptors of the immune system, see, for example, Springer (Springer, Nature, 346:425-434 (1990)) and Osborn (Osborn, Cell, 62:3-6 (1990)).
Inflammatory brain disorders, such as multiple sclerosis (MS) and meningitis, are examples of central nervous system disorders in which the endothelium/leukocyte adhesion mechanism results in destruction to otherwise healthy brain tissue. Large numbers of leukocytes migrate across the blood brain barrier (BBB) in subjects with these inflammatory diseases. The leukocytes release toxic mediators that cause extensive tissue damage resulting in impaired nerve conduction and paralysis.
In other organ systems, tissue damage also occurs via an adhesion mechanism resulting in migration or activation of leukocytes. For example, it has been shown that the initial insult following myocardial ischemia to heart tissue can be further complicated by leukocyte entry to injured tissue causing still further insult (Vedder, et al., Surgery, 106:509 (1989)). Other inflammatory conditions mediated by an adhesion mechanism include asthma (Pretolani, et al., J. Exp. Med., 180:795 (1994); Abraham, et al., Clin. Invest., 93:776 (1994); Mulligan, et al., Immunology, 150:2407 (1993)), Alzheimer's disease, atherosclerosis (Cybulsky, et al., Science, 251:788 (1991); Li, et al., Atheroscler. Thromb., 13:197 (1993)), AIDS dementia (Sasseville, et al., Am. J. Path., 144:27 (1994)), diabetes (Yang, et al., Proc. Nat. Acad. Science (USA), 90:10494 (1993); Burkly, et al., Diabetes, 43:526 (1994); Baron, et al., J. Clin. Invest., 93:1700 (1994)), inflammatory bowel disease (Hamann, et al., Immunology, 152:3238 (1994)), multiple sclerosis (Yednock, et al., Nature, 356:63 (1992); Baron, et al., J. Exp. Med., 177:57 (1993)), rheumatoid arthritis (van Dinther-Janssen, et al., Annals. Rheumatic Dis., 52:672 (1993); Elices, et al., J. Clin. Invest., 93:405 (1994); Postigo, et al., J. Clin. Invest., 89:1445 (1991)), tissue transplantation (Paul, et al., Transpl. Proceed., 25:813 (1993), and tumor metastasis (Okahara, et al., Can. Res., 54:3233 (1994); Paavonen, et al., Int. J. Can., 58:298 (1994); Schadendorf, et al., J. Path., 170:429 (1993)).
Because of the significance of VLA-4 in inflammatory and autoimmune conditions, it is desirable to test for the presence of VLA-4 in biological samples and for compounds which inhibit cell adhesion.
Individually, each receptor/ligand interaction is rapidly reversible; however, during the process of cell adhesion, multiple .alpha..sub.4 .beta..sub.1 integrin receptors on one cell engage multiple VCAM-1 ligands on another cell, and together provide a strong and stable adhesive bond. In order to prevent cell adhesion, small molecule inhibitors of .alpha..sub.4 .beta..sub.1 integrin must achieve a high degree of receptor occupancy for disruption of a significant number of these adhesive interactions. Furthermore, due to the multivalency of the adhesive interaction, inhibitory compounds exhibit a very steep titration curve, since inhibition begins with 85-90% receptor occupancy and is complete when 95-100% of the receptors are occupied. With such a narrow dynamic range there is considerable assay to assay variation in cell-based adhesion studies. An assay which can detect the presence of a single VCAM-1 molecule with a single receptor and thus prevent assay to assay variation is desired.
N-substituted 3,4-diamino-3-cyclobutene-1,2-dione derivatives have been taught. Japanese Patent JP05229999 A2 930907 discloses cyclobutenediones which are symmetrically disubstituted with .alpha.-amino acids.
U.S. Pat. No. 5,168,103 issued Dec. 1, 1992, and assigned to American Home Products, describes cyclobutenedione derivatives having formula (2) ##STR2## where A is alkylene or alkenylene. These compounds are taught to be useful as N-methyl-D-aspartate antagonists.